RasGAP derived peptide for selectively killing cancer cells

ABSTRACT

The present invention relates to a peptide consisting in the N2 sequence of the RasGAP protein, a fragment thereof, or a variant thereof which enhances the ability of a drug to selectively kill cells. Furthermore, it relates to a pharmaceutical composition comprising as an active substance a pharmaceutically effective amount of the peptide.

RELATED APPLICATIONS

This application is a 35 U.S.C. §371 filing of International ApplicationNumber PCT/IB2004/002165 which was filed Jun. 30, 2004, which claimspriority to U.S. Provisional Application No. 60/483,691, filed on Jun.30, 2003. The contents of the aforementioned applications are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a peptide which enhances the ability ofa drug to kill cells selectively in cancer cells. Furthermore, itrelates to a pharmaceutical composition comprising as an activesubstance a pharmaceutically effective amount of at least one of saidpeptide.

BACKGROUND OF THE INVENTION

Tumors are diverse and heterogeneous, but all share the ability toproliferate without control. Deregulated cell proliferation coupled withsuppressed apoptotic sensitivity constitutes a minimal requirement uponwhich tumor evolution occurs.

Apoptosis is the process by which cells enter programmed cell death, avital phenomenon that takes place during development, and is essentialfor the maintenance of homeostasis. The biochemical event that isbelieved to irreversibly commit a cell to apoptosis is the activation ofcaspases (cysteine proteases cleaving after aspartic residues). Cellsundergoing apoptosis display characteristic morphological andbiochemical changes, including membrane blebbing, cell rounding,chromatin condensation, DNA cleavage, expression of apoptotic markers atthe cell surface and inhibition of anti-apoptotic signaling pathways.All these events can be blocked by specific caspase inhibitors. It isthus the cleavage of the caspase substrate that is responsible for most,if not all, of the characteristic changes observed during apoptosis.

The execution phase of apoptosis is triggered when caspase substrates ina cell are cleaved. Dozens of caspase substrates have been identifiedand the list is growing steadily (Earnshaw W. C. et al., “Mammaliancaspases: structure, activation, substrates, and functions duringapoptosis” Annu. Rev. Biochem. 68, 383, 1999). Once cleaved, caspasesubstrates mediate the biochemical and morphological events observedduring apoptosis such as amplification of the activation of caspases,DNA fragmentation, nuclear breakdown, etc.

Furthermore, Mitogen-activated protein kinase (MAPK) pathways have beenshown to regulate apoptosis in a positive or negative manner (Jarpe M.B. et al., “Anti-apoptotic versus pro-apoptotic signal transduction:checkpoints and stop signs along the road to death” Oncogene, 17, 1475,1998; Widmann C. et al, “Mitogen-activated protein kinase: conservationof a three-kinase module from yeast to human” Physiol. Rev. 79, 143,1999). This could explain why the apoptotic caspases target some of thesignaling proteins that regulate MAPK and/or are components of MAPKpathways (Widmann C. et al., “Caspase-dependent cleavage of signalingproteins during apoptosis. A turn-off mechanism for anti-apoptoticsignals” J. Biol. Chem., 273, 7141, 1998). These proteins include MEKK1,PAK2, Mst1 and RasGAP.

Recently, Yang and Widmann, (Yang J.-Y. and Widmann C., “Antiapoptoticsignaling generated by caspase-induced cleavage of RasGAP” Mol. Cell.Biol., 21, 5346, 2001; “A subset of caspase substrates functions as theJekyll and Hyde of apoptosis” Eur. Cytokine Netw., 13, 387, 2002a; “TheRasGAP N-terminal fragment generated by caspase cleavage protects cellsin a Ras/PI3K/Akt-dependent manner that does not rely on NFkappa B” J.Biol. Chem., 277, 14641, 2002b), have demonstrated that RasGAP, aregulator of Ras and Rho GTP-binding proteins, is an unconventionalcaspase substrate because it can induce both anti- and pro-apoptoticsignals, depending on the extent of its cleavage by caspases. They haveshown that at low levels of caspase activity, RasGAP is cleaved atposition 455, generating an N-terminal sequence (sequence N) and aC-terminal sequence (sequence C).

Sequence C, but not full-length RasGAP, induced a strong apoptoticresponse in HeLa cells as assessed by its ability to induce theappearance of pycnotic nuclei, activation of caspase 3, and cleavage ofPARP.

In the same study, the authors have also shown that sequence N, ratherthan promoting cell death, appears to be a general blocker of apoptosisdownstream of caspase activation. At higher levels of caspase activity,the ability of sequence N to counteract apoptosis is suppressed when itis cleaved at position 157. This latter cleavage event generates twosequences, N1 and N2, that in contrast to sequence N, have been shown toseitizises cells which can develop high caspase activities towardapoptosis induced by cisplatin, a drug used in chemotherapy to treatcancers.

However, it has been shown in Leblanc et al (Leblanc V. et al.,“Ras-GTPase activating protein inhibition specifically induces apoptosisof tumour cells” Oncogene, 18, 4884, 1999) that injection of amonoclonal antibody directed against the SH3 domain of the N2 sequenceof RasGAP in order to inhibit this protein specifically inducesapoptosis in cancer cells. It is also know from patent applicationWO99/65947 (Parker et al.) that monoclonal antibodies directed against aRasGAP SH3-domain-binding protein, G3BP, induce apoptosis in cancercells in which G3BP is specifically overexpressed.

These results seem to indicate that the RasGAP pathway regulatinggrowth, through the RasGAP SH3 domain, is essential for some cancercells to survive. These findings seem to be in contrast with the resultsobtained by Yang and Widmann thus leading to the conclusion that theRasGAP SH3 domain has a quite ambivalent function in the induction andregulation of apoptosis in cells.

Chemotherapy, alone or in combination with other treatments (e.g.radiotherapy), are currently one of the most common and efficienttherapeutical tool to treat cancers. The efficacy of the drugs used inchemotherapy to treat cancers relies on their ability to kill cancercells. There is, however, a limitation in the use of these drugs thatcomes from the fact that they can also adversely affect normal cells,non cancer cells, since they not only induce a strong stimulation ofcaspases in cancer cells but also in normal cells, non cancer cells,especially those cells that divide quickly.

Therefore, the challenge for the clinicians is to choose the doses ofdrugs that are high enough to eliminate the tumors but not so high as toinduce severe side effects in the patients such as hair loss, nausea andvomiting, cardiac toxicity and secondary cancers.

Improving the selectivity of drugs towards cancer cells would obviouslyincrease the efficacy of chemotherapeutic treatments thereby enablinglower doses of drugs. This will also result in reducing as far aspossible the severe above-listed side effects.

Therefore, the object of the present invention is to provide an improvedapproach, in combination with a drug, for the treatment or prevention ofcancers, which does not have the above-mentioned drawbacks.

SUMMARY OF THE INVENTION

This object has been achieved by providing a peptide consistingessentially of the N2 sequence of the RasGAP protein, a fragmentthereof, or a variant thereof, which enhances the ability of a drug tokill selectively cancer cells.

Furthermore, the invention provides a purified and isolated nucleic acidsequence encoding the peptide, an expression vector comprising at leastone copy of purified and isolated nucleic acid sequence and a eukaryoticor prokaryotic host cell containing the peptide, the isolated andpurified nucleic acid sequence and/or the expression vector.

The invention further provides a pharmaceutical composition comprisingas an active substance a pharmaceutically effective amount of at leastone peptide according to the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage of apoptosis induction by various drugs inHeLa cells transfected either with Fragment N2 or an empty pcDNAplasmid.

HeLa cells (2×10⁶) were plated in 10-cm-diameter Petri dishes,transfected with 1 μg of GFP-expressing plasmid (to label thetransfected cells) together with 2 μg of an empty pcDNA3 plasmid or 2 μgof a pcDNA3 vector encoding fragment N2. One day following thetransfection, the cells were incubated for 24 hours with the indicatedconcentrations of cisplatin, adriamycin or mitoxantrone. The number ofGFP-positive cells displaying pycnotic nucleus was then scored.

The results correspond to the mean±standard deviation of threeindependent determinations. Asterisks denote significant differencesbetween control cells and cells treated with cisplatin (*, p<0.05; **,p<0.01; ***, p<0.001).

FIG. 2A is a schematic representation of the different constructs usedin this study.

SH represents the Src homology domain.

FIG. 2B shows the percentage of apoptosis induction by cisplatin in HeLacells transfected with plasmids encoding the constructs described inFIG. 2A.

HeLa cells were transfected as described in FIG. 1 with plasmidsendoding the constructs described in FIG. 2A. HeLa cells were thentreated or not with 0.15 μM cisplatin and the extent of apoptosis wasdetermined 20 hours later. The results correspond to the mean±standarddeviation of three independent determinations. Asterisks denote asignificant difference between the cells treated with 0.15 μM cisplatinand the cells without treatment (**, p<0.01; ***, p<0.001).

FIG. 3 shows phase contrast and epifluorescence images of ilve cellsincubated with FITC-labelled TAT-RasGAP₃₁₇₋₃₂₆ peptide.

The following cell lines (HeLa, U2OS, H-Meso1, MCF-7, HaCat andHUV-EC-C) were incubated 3 hours at 37° C., 5% CO₂ in culture mediumwith 20 μM FITC-labelled TAT-RasGAP₃₁₇₋₃₂₆ peptide and then washed threetimes with culture medium.

FIG. 4A shows the percentage of apoptosis induction by various drugs intwo non-cancer cells treated or not with TAT-RASGAP₃₁₇₋₃₂₆.

Two non-cancer cell lines (HaCat and HUV-EC-C) were incubated withincreasing concentrations of cisplatin, adriamycin and mitoxantrone inthe absence or in the presence of 20 μM TAT-RaasGA-P₃₁₇₋₃₂₆. The extentof apoptosis was scored 20 hours later.

FIG. 4B shows the percentage of apoptosis induction by various drugs infour cancer cell lines treated or not with TAT-RASGAP₃₁₇₋₃₂₆.

Four cancer cell lines (HeLa, U2OS, MCF-7, and H-Meso1) were plated in6-wells plates and treated with the indicated concentrations ofcisplatin, adriamycin and mitoxantrone in the absence or in the presenceof either 20 μM MV-TAT₄₈₋₅₇ Or TAT-RaSGAP₃₁₇₋₃₂₆ peptides for 20 hours.The number of cells displaying pycnotic nucleus was then scored. Theresults correspond to the mean±standard deviation of three independentdeterminations. Asterisks denote significant differences between thegenotoxin-treated cells incubated with TAT-RaSGAP₃₁₇₋₃₂₆ and those leftuntreated or incubated with the HIV-TAT₄₈₋₅₇ peptide (**, p<0.01; ***,p<0.001).

FIG. 5A shows the NFκB activity in U2OS cells treated with cisplatin andin the presence of HIV-TAT₄₈₋₅₇ or TAT-RaSGAP₃₁₇₋₃₂₆ peptides

U2OS Cells (1×10⁵) were plated in 6 well plates and transfected with 1μg of a firefly luciferase reporter plasmid for NFκB activity and 0.1 μgof a plasmid encoding the Renilla luciferase. The cells were treated oneday later with the indicated concentrations of cisplatin in the absenceor in the presence of 20 μM HV-TAT₄₈₋₅₇ or TAT-RaSGAP₃₁₇₋₃₂₆ peptidesduring 20 hours. The data represent the firefly luciferase activitynormalized to the Renilla luciferase activity and expressed as foldincrease of the basal NFκB activity obtained in control untreated cells.The results correspond to the mean±standard deviation of threeindependent determinations. Asterisks denote significant differencesbetween the indicated conditions (**, p<0.01; ***, p<0.001).

FIG. 5B shows the NFκB activity in U2OS cells treated with cisplatin andin the presence of HIV-TAT₄₈₋₅₇ peptide and IκBαΔN2 or TAT-RasGAP₃₁₇₋₃₂₆peptide and IκBαΔN2.

U2OS cells were transfected with 1 μg of a firefly luciferase reporterplasmid for NFκB activity, 0.1 μg of a plasmid encoding the Renillaluciferase, 0.5 μg of a GFP-expressing plasmid (to label the transfectedcells), 1 μg of a plasmid encoding IκBαΔN2 that inhibit the NFκB pathwayor with 1 μg an empty pcDNA3 vector. The cells were incubated one daylater with increasing concentrations of cisplatin in the presence of 20μM HIV-TAT₄₈₋₅₇ or 20 μM TAT-RasGAP₃₁₇₋₃₂₆ for an additional 20 hourperiod. The cells were then lysed and the NFκB activity assessed asdescribed in panel A, except that the results were expressed as foldincrease of the NFκB activity detected in cells incubated with thecontrol HV-TAT₄₈₋₅₇ peptide.

FIG. 5C shows the percentage of apoptosis of transfected cells of FIGS.5A and B.

Alternatively, the number of GFP-positive cells displaying pycnoticnucleus was determined. The results correspond to the mean±standarddeviation of three independent determinations.

FIG. 6 represents western blots and percentage of JNK phosphorylationsof U2OS cells transfected with either HIV-TAT₄₈₋₅₇ or TAT-RasGAP₃₁₇₋₃₂₆peptides and treated with cisplatin.

U2OS cells (2×10⁵) were plated in 6 well plates and treated for theindicated periods of time with the indicated combinations of theHW-TAT₄₈₋₅₇ or TAT-RasGAP₃₁₇₋₃₂₆ peptides (at a 20 μM concentration) andcisplatin (at a 30 μM concentration). Positive controls for JNK and p38activation were obtained following stimulation of the cells with 1 μg/mlanisomycine during 3 hours and with 0.5 M sorbitol for 30 min,respectively. The quantitations depicted under the Western blots wereperformed on the 12-hour bands and normalized against the positivecontrols. The results correspond to the mean±standard deviation of threeindependent experiments.

DETAILED DESCRIPTION OF THIE INVENTION

As used herein, the terms “peptide”, “protein”, “polypeptide”,“polypeptidic” and “peptidic” are used interchangeably to designate aseries of amino acid residues connected to the other by peptide bondsbetween the alpha-amino and carboxy groups of adjacent residues.

RasGAP, a regulator of Ras and Rho GTP-binding proteins, is anunconventional caspase substrate because it can induce both anti- andpro-apoptotic signals, depending on the extent of its cleavage bycaspases. At low levels of caspases, RasGAP is cleaved at position 455,generating an N-terminal fragment (fragment N, of about 56 kD) and aC-terminal fragment (fragment C, of about 64 kD). Fragment N appears tobe a general blocker of apoptosis downstream of caspase activation (YangJ.-Y. and Widmann C., Mol. Cell. Biol., 21, 5346, 2001 and J. Biol.Chem., 277, 14641, 2002b). At high levels of caspase activity, fragmentN is further cleaved at position 157 thus generating two fragments, N1(amino acids 1 to 157) and N2 (amino acids 158 to 455).

By “cancer cell” is meant a cell arising in an animal in vivo which iscapable of undesired and unregulated cell growth or abnormal persistenceor abnormal invasion of tissues. In vitro this term also refers to acell line that is a permanently immortalized established cell culturethat will proliferate indefinitely and in an unregulated manner givenappropriate fresh medium and space.

The term “drug” refers to drugs which are able to kill mammalian cells,preferably human cells. There are several classes of drugs of differentorigin and with different modes of action.

Drugs of the present invention concern agents which are derived from, orwhich beneficially modulate host biological processes. Interferons,tumor growth factors, tumor necrosis factors, growth factors such asGM-CSF and G-CSF and interleukins such as interleukin-2, interleukin-6,interleukin-7 and interleukin-12 are examples of such biological drugscurrently used in cancer therapeutics.

A drug of the present invention can concern as well agents which damageDNA and/or prevent cells from multiplying, such as genotoxins.Genotoxins can be selected from the group comprising alkylating agents,antimetabolites, DNA cutters, DNA binders, topoisomerase poisons andspindle poisons.

Examples of alkylating agents are lomustine, carmustine, streptozocin,mechlorethamine, melphalan, uracil nitrogen mustard, chlorambucil,cyclosphamide, iphosphamide, cisplatin, carboplatin, mitomycin,thiotepa, dacarbazin, procarbazine, hexamethyl melamine, triethylenemelamine, busulfan, pipobroman, mitotane and other platine derivatives.

An example of DNA cutters is bleomycin.

Toposiomerases poisons can be selected from the group comprisingtopotecan, irinotecan, camptothecin sodium salt, daorubicin,doxorubicin, idarubicin, mitoxantrone teniposide, adriamycin andetoposide.

Examples of DNA binders are dactinomycin and mithramycin whereas spindlepoisons can be selected among the group comprising vinblastin,vincristin, navelbin, paclitaxel and docetaxel.

As drug as well antimetabolites can be used which can be selected amongthe following coumpounds: methotrexate, trimetrexate, pentostatin,cytarabin, ara-CMP, fludarabine phosphate, hydroxyurea, fluorouracyl,floxuridine, chlorodeoxyadenosine, gemcitabine, thioguanine and6-mercaptopurine.

Preferably a genotoxin, more preferably cisplatin, mitoxantrone andadriamycin are used as drug in the present invention.

These drugs can be used alone or in combination with one another. Incase more than one drug is used the determination of a usefulcombination of drugs is well within the capabilities of those skilled inthe art, and will depend e. g. on the cancer cells to kill.

The term “enhancing” as used herein refers to the capacity of a peptideto increase the effect of a drug to kill cells. This capacity can bemeasured in vitro by, for example, measuring the percentage of apoptosisof cells containing a peptide and treated with at least one drug byscoring the number of cells displaying pycnotic nuclei (a marker ofapoptotic cells). Typically, the results are compared to those fromdrug-treated cells that do not contain said peptide. A peptide thatleads to a two fold or more increase of apoptosis in cells at a givenconcentration or that decreases by at least two-fold the dose of a drugto induce a given apoptotic response, will be considered as enhancingthe ability of a drug to kill cells.

As used herein, by the term “selectively” is meant that the peptide ofthe invention enhances the ability of a drug to kill cells at a givenconcentration, specifically in cancer cells but surprisingly not in noncancer cells.

Concentration ranges of a drug in vitro in which the peptide enhancesthe ability of a drug to kill cells selectively in cancer cells depend,usually, on the drug used. For example, in case a genotoxin is used,usually the concentration of the drug in vitro is between 0.1 to 100 μM,preferably between 0.15 to 30 μM.

The N2 sequence of the RasGAP protein is preferably derived from humanand refers to a 36 kD protein consisting of 297 amino acids whichencompasses two SH2 and one SH3 domain as shown in FIG. 2A.

In general, Src homology 2 (SH2) domains are involved in recognition ofphosphorylated tyrosine whereas Src homology 3 (SH3) domains are oftenindicative of a protein involved in signal transduction related tocytoskeletal organisation.

“Fragment” refers to a sequence containing less amino acids in lengththan the N2 sequence of the RasGAP protein. This sequence can be used aslong as it exhibits the same properties as the native sequence fromwhich it derives. Preferably this sequence contains less than 90%,preferably less than 60%, in particular less than 30% amino acids inlength than the respective N2 sequence of the RasGAP protein.

The present invention also includes a variant of the N2 sequence of theRasGAP protein. The term “variant” refers to a peptide having an aminoacid sequence that differ to some extent from a native sequence peptide,that is an amino acid sequence that vary from the native sequence byconservative amino acid substitutions, whereby one or more amino acidsare substituted by another with same characteristics and conformationalroles. The amino acid sequence variants possess substitutions,deletions, and/or insertions at certain positions within the amino acidsequence of the native amino acid sequence. Conservative amino acidsubstitutions are herein defined as exchanges within one of thefollowing five groups:

-   I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser,    Thr, Pro, Gly-   II. Polar, positively charged residues: His, Arg, Lys-   III. Polar, negatively charged residues: and their amides: Asp, Asn,    Glu, Gln-   IV. Large, aromatic residues: Phe, Tyr, Trp-   V. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys.

The N2 sequence, as well as a fragment and a variant thereof can beprepared by a variety of methods and techniques known in the art such asfor example chemical synthesis or recombinant techniques as described inManiatis et al. 1982, Molecular Cloning, A laboratory Manual, ColdSpring Harbor Laboratory.

Preferably, the fragment of the N2 sequence of the RasGAP proteincomprises the amino acid sequence of the SH3 domain of the N2 sequence,a part thereof, or a variant thereof.

Applicants, surprisingly, have characterized a shorter sequence of theN2 sequence of the RasGAP protein that still enhances the ability of adrug to kill selectively cancer cells and that has also the advantage tobe more easily synthesized. In order to assess whether such a partthereof could be isolated, applicants have generated a series oftruncated versions of fragment N, as shown in FIG. 2 A and determinedtheir ability to potentiate drug induced apoptosis in a cancer cell line(FIG. 2 B). These parts of the N2 sequence have been cloned into avector and transfected into a cancer cell line (HeLa).

Applicants have shown that HeLa cells transfected with an emptyconstruct or with a construct encoding the SH2 domain of N2 only do notenhance cisplatin-induced death. In contrast, cells expressing theconstructs containing the SH3 domain had an increased enhancement ofcisplatin-induced apoptosis as shown in FIG. 2B

Applicants have then generated progressive truncations in the SH3 domainin an attempt to identify a minimal enhancing sequence. All theseconstructs or parts of the N2 sequence (FIG. 2A), including the shortestone (317-326) that codes for a 10 amino acid long peptide, potentiatedthe ability of cisplatin to kill HeLa cells (FIG. 2B). These resultsshow that the cell-death enhancing property of fragment N2 does notrequire a complete SH3 domain but is mediated by a part of the SH3domain such as a short peptidic sequence.

The part of the SH3 domain or the variant thereof contains preferablyless than or equal to 70, more preferably less than or equal to 30, mostpreferably less than or equal to 10 amino acids of the amino acidsequence of the SH3 domain.

In particular encompassed by the present invention is a part of the SH3domain which consists in the amino acid sequences encoded by the DNAsequences of Table 1:

TABLE 1 Amino Acid Sequences Name DNA sequences sequences SEQ ID N° 1RasGAP_(284–351) gaagatagaaggcgtgtacgagctattctacctta EDRRRVRAILPYTKVcacaaaagtaccagacactgatgaaataagtttct PDTDEISFLKGDMFItaaaaggagatatgttcattgttcataatgaatta VHNELEDGWMWVTNLgaagatggatggatgtgggttacaaatttaagaac RTDEQGLIVEDLVEEagatgaacaaggccttattgttgaagacctagtag VGREEDPHEGKIWFHaagaggtgggccgggaagaagatccacatgaagga GKISKQEAaaaatatggttccatgggaagatttccaaacagga agct SEQ ID N° 2 RasGAP_(284–341)gtacgagctattctaccttacacaaaagtaccaga RVRAILPYTKVPDTDcactgatgaaataagtttcttaaaaggagatatgt EISFLKGDMFIVHNEtcattgttcataatgaattagaagatggatggatg LEDGWMWVTNLRTDEtgggttacaaatttaagaacagatgaacaaggcct QGLIVEDLVEEVGREtattgttgaagacctagtagaagaggtgggccggg EDPHEGKIWaagaagatccacatgaaggaaaaatatgg SEQ ID N° 3 RasGAP_(284–336)gtacgagctattctaccttacacaaaagtaccaga RVRAILPYTKVPDTDcactgatgaaataagtttcttaaaaggagatatgt EISFLKGDMFIVHNEtcattgttcataatgaattagaagatggatggatg LEDGWMWVTNLRTDEtgggttacaaatttaagaacagatgaacaaggcct QGLIVEDLVEEVGRtattgttgaagacctagtagaagaggtgggccgg SEQ ID N° 4 RasGAP_(317–326)tggatgtgggttacaaatttaagaacagat WMWVTNLRTD

In case the part of the SH3 domain of the N2 sequence is SEQ ID NO: 4(RasGAP₃₁₇₋₃₂₆) then the resulting amino acid sequence encoded by saidSEQ ID NO: 4 in human is WMWVTNLRTD (SEQ ID NO: 8). A comparison betweenthe different species revealed that there are different amino acids,which are conserved among the species as shown in table 2.

TABLE 2 Amino acid sequences Species of RasGAP_(317–326) HumanWMWVTNLRTD Bos taurus WMWVTNLRTD Mouse WMWVTNLRTD Rattus norvegicusWMWVTNLRTD Anopheles WLWVTAHRTG Drosophila WLWVTAHRTG Alignment W x WVTxx RT x

Conserved amino acids among the species are represented as boldunderlined type residues whereas the X correspond to amino acid residuesthat can be changed by conservative, or non-conservative amino acidsubstitutions, without impairing the inventive properties of these 10amino acid parts of the SH3 domain of N2.

These peptidic variants of this 10 amino acid part of the human SH3domain of N2, and in particular the alignment sequence WXWVTXXRTX (SEQID NO: 14), are also encompassed by the present invention and they referto peptides having an amino acid sequence that differ to some extentfrom the native sequence peptide, that is the amino acid sequence thatvary from the native sequence WMWVTNLRTD (SEQ ID NO: 8) by conservativeor non-conservative amino acid substitutions, whereby one or more aminoacid residues are substituted by another with same characteristics andconformational roles.

Usually, the peptide consisting essentially of the N2 sequence of theRasGAP protein, a fragment thereof, or a variant thereof as disclosed inthe present invention is conjugated to an agent which increases theaccumulation of the peptide in a cell.

Such an agent can be a compound which induces receptor mediatedendocytose such as for example the membrane transferrin receptormediated endocytosis of transferrin conjugated to therapeutic drugs(Qian Z. M. et al., “Targeted drug delivery via the transferrinreceptor-mediated endocytosis pathway” Pharmacological Reviews, 54, 561,2002) or a cell membrane permeable carrier which can, be selected e. g.among the group of fatty acids such as decanoic acid, myristic acid andstearic acid, which have already been used for intracellular delivery ofpeptide inhibitors of protein kinase C (Ioannides C. G. et al.,“Inhibition of IL-2 receptor induction and IL-2 production in the humanleukemic cell line Jurkat by a novel peptide inhibitor of protein kinaseC” Cell Immunol., 131, 242, 1990) and protein-tyrosine phosphatase (KoleH. K. et al., “A peptide-based protein-tyrosine phosphatase inhibitorspecifically enhances insulin receptor function in intact cells” J.Biol. Chem. 271, 14302, 1996) or among peptides. Preferably, cellmembrane permeable carriers are used, more preferably a cell membranepermeable carrier peptide is used.

In case the cell membrane permeable carrier is a peptide then it willpreferably be an arginine rich peptide. It has been recently shown inFutaki et al. (Futaki S. et al., “Arginine-rich peptides. An abundantsource of membrane-permeable peptides having potential as carriers forintracellular protein delivery” J. Biol. Chem., 276, 5836, 2001), thatthe number of arginine residues in a cell membrane permeable carrierpeptide has a significant influence on the method of internalization andthat there seems to be an optimal number of arginine residues for theinternalization, preferably they contain more than 6 arginines.

The peptide of the invention is usually conjugated to the cell membranepermeable carrier by a spacer. In this case the cell membrane permeablecarrier is preferably a peptide.

Usually arginine rich peptides are selected from the group comprisingthe HIV-TAT₄₈₋₅₇ peptide, the FHV-coat₃₅₋₄₉ peptide, the HTLV-II Rex₄₋₁₆peptide and the BMV gag₇₋₂₅ peptide. Preferably, the arginine richpeptide is HIV-TAT₄₈₋₅₇ peptide.

In case the HIV-TAT₄₈₋₅₇ peptide is conjugated to a RasGAP sequence,such as for example RasGAP₃₁₇₋₃₂₆, then two glycine residues areinserted between the TAT and RasGAP sequences as spacer to allowflexibility.

Since an inherent problem with native peptides (in L-form) isdegradation by natural proteases, the peptide of the invention may beprepared to include D-forms and/or “retro-inverso isomers” of thepeptide.

In this case, retro-inverso isomers of short fragments and variants ofthe peptide of the invention are prepared.

Protecting the peptide from natural proteolysis should thereforeincrease the effectiveness of the specific heterobivalent orheteromultivalent compound. A higher biological activity is predictedfor the retro-inverso containing peptide when compared to thenon-retro-inverso containing analog owing to protection from degradationby native proteinases. Furthermore they have been shown to exhibit anincreased stability and lower immunogenicity (Sela M. and Zisman E.,“Different roles of D-amino acids in immune phenomena” FASEB J. 11, 449,1997).

Retro-inverso peptides are prepared for peptides of known sequence asdescribed for example in Sela and Zisman, (1997).

By “retro-inverso isomer” is meant an isomer of a linear peptide inwhich the direction of the sequence is reversed and the chirality ofeach amino acid residue is inverted; thus, there can be no end-groupcomplementarity.

Also encompassed by the present invention are modifications of thepeptide (which do not normally alter primary sequence), including invivo or in vitro chemical derivitization of peptides, e. g., acetylationor carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a peptideduring its synthesis and processing or in further processing steps, e.g., by exposing the peptide to enzymes which affect glycosylation e. g.,mammalian glycosylating or deglycosylating enzymes. Also included aresequences which have phosphorylated amino acid residues, e. g.,phosphotyrosine, phosphoserine, or phosphothreonine.

The invention also includes analogs in which one or more peptide bondshave been replaced with an alternative type of covalent bond (a “peptidemimetic”) which is not susceptible to cleavage by peptidases. Whereproteolytic degradation of the peptides following injection into thesubject is a problem, replacement of a particularly sensitive peptidebond with a noncleavable peptide mimetic will make the resulting peptidemore stable and thus more useful as an active substance. Such mimetics,and methods of incorporating them into peptides, are well known in theart.

Also useful are amino-terminal blocking groups such ast-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl,adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl,methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl.Blocking the charged amino- and carboxy-termini of the peptides wouldhave the additional benefit of enhancing passage of the peptide throughthe hydrophobic cellular membrane and into the cell.

When recombinant techniques are employed to prepare a peptide consistingessentially of the N2 sequence of the RasGAP protein, a fragmentthereof, or a variant thereof, in accordance with the present invention,nucleic acid sequences encoding the polypeptides are preferably used.With regard to the method to practise recombinant techniques, see forexample, Maniatis et al. 1982, Molecular Cloning, A laboratory Manual,Cold Spring Harbor Laboratory and commercially available methods.

Therefore the present invention also relates to a purified and isolatednucleic acid sequence encoding a peptide consisting essentially of theN2 sequence of the RasGAP protein, a fragment thereof, or a variantthereof as described above.

“A purified and isolated nucleic acid or nucleic acid sequence” refersto the state in which the nucleic acid sequence encoding the peptide ofthe invention, or nucleic acid encoding such peptide consistingessentially of the N2 sequence of the RasGAP protein, a fragmentthereof, or a variant thereof will be, in accordance with the presentinvention.

A purified and isolated nucleic acid or nucleic acid sequenceencompassed by the present invention might be DNA, RNA, or DNA/RNAhybrid.

DNA which can be used herein is any polydeoxynuclotide sequence,including, e.g. double-stranded DNA, single-stranded DNA,double-stranded DNA wherein one or both strands are composed of two ormore fragments, double-stranded DNA wherein one or both strands have anuninterrupted phosphodiester backbone, DNA containing one or moresingle-stranded portion(s) and one or more double-stranded portion(s),double-stranded DNA wherein the DNA strands are fully complementary,double-stranded DNA wherein the DNA strands are only partiallycomplementary, circular DNA, covalently-closed DNA, linear DNA,covalently cross-linked DNA, cDNA, chemically-synthesized DNA,semi-synthetic DNA, biosynthetic DNA, naturally-isolated DNA,enzyme-digested DNA, sheared DNA, labeled DNA, such as radiolabeled DNAand fluorochrome-labeled DNA, DNA containing one or more non-naturallyoccurring species of nucleic acid.

DNA sequences that encode a peptide consisting essentially of the N2sequence of the RasGAP protein, a fragment thereof, or a variantthereof, can be synthesized by standard chemical techniques, forexample, the phosphotriester method or via automated synthesis methodsand PCR methods.

The purified and isolated DNA sequence encoding a peptide consistingessentially of the N2 sequence of the RasGAP protein, a fragmentthereof, or a variant thereof, according to the invention may also beproduced by enzymatic techniques. Thus, restriction enzymes, whichcleave nucleic acid molecules at predefined recognition sequences can beused to isolate nucleic acid sequences from larger nucleic acidmolecules containing the nucleic acid sequence, such as DNA (or RNA)that codes for a peptide consisting essentially of the N2 sequence ofthe RasGAP protein, a fragment thereof, or a variant thereof.

Encompassed by the present invention is also a nucleic acid in the formof a polyribonucleotide (RNA), including, e.g., single-stranded RNA,cRNA, double-stranded RNA, double-stranded RNA wherein one or bothstrands are composed of two or more fragments, double-stranded RNAwherein one or both strands have an uninterrupted phosphodiesterbackbone, RNA containing one or more single-stranded portion(s) and oneor more double-stranded portion(s), double-stranded RNA wherein the RNAstrands are fully complementary, double-stranded RNA wherein the RNAstrands are only partially complementary, covalently crosslinked RNA,enzyme-digested RNA, sheared RNA, MnRNA, chemically-synthesized RNA,semi-synthetic RNA, biosynthetic RNA, naturally-isolated RNA, labeledRNA, such as radiolabeled RNA and fluorochrome-labeled RNA, RNAcontaining one or more non-naturally-occurring species of nucleic acid.

Preferably used as nucleic acid is a purified and isolated DNA sequenceselected from the group comprising SEQ ID No 1, SEQ ID No 2, SEQ ID No3, or SEQ ID No 4.

The present invention also includes variants of the aforementionedsequences, that is nucleotide sequences that vary from the referencesequence by conservative nucleotide substitutions, whereby one or morenucleotides are substituted by another with same characteristics.

The invention also encompasses allelic variants of the disclosedpurified and isolated nucleic sequence; that is, naturally-occurringalternative forms of the isolated and purified nucleic acid that alsoencode peptides that are identical, homologous or related to thatencoded by the purified and isolated nucleic sequences. Alternatively,non-naturally occurring variants may be produced by mutagenesistechniques or by direct synthesis.

The aforementioned purified and isolated nucleic acid sequence encodinga peptide consisting essentially of the N2 sequence of the RasGAPprotein, a fragment thereof, or a variant thereof, may further comprisea nucleotide sequence encoding a cell membrane permeable carrierpeptide.

Yet another concern of the present invention is to provide an expressionvector comprising at least one copy of the isolated and purified nucleicacid sequence encoding a peptide consisting essentially of the N2sequence of the RasGAP protein, a fragment thereof, or a variant thereofas described above. Preferably the isolated and purified nucleic acidsequence encoding a peptide of the invention is DNA.

As used herein, “vector”, “plasmid” and “expression vector” are usedinterchangeably, as the plasmid is the most commonly used vector form.

The vector may further comprise a nucleotide sequence encoding a cellmembrane permeable carrier peptide in accordance with the invention. Thechoice of an expression vector depends directly, as it is well known inthe art, on the desired functional properties, e.g., peptide expressionand the host cell to be transformed or transfected.

Additionally, the expression vector may further comprise a promoteroperably linked to the purified and isolated DNA sequence. This meansthat the linked isolated and purified DNA sequence encoding the peptideof the present invention is under control of a suitable regulatorysequence which allows expression, i.e. transcription and translation ofthe inserted isolated and purified DNA sequence.

As used herein, the term “promoter” designates any additional regulatorysequences as known in the art e.g. a promoter and/or an enhancer,polyadenylation sites and splice junctions usually employed for theexpression of the polypeptide or may include additionally one or moreseparate targeting sequences and may optionally encode a selectablemarker. Promoters which can be used provided that such promoters arecompatible with the host cell are e.g promoters obtained from thegenomes of viruses such as polyoma virus, adenovirus (such as Adenovirus2), papilloma virus (such as bovine papilloma virus), avian sarcomavirus, cytomegalovirus (such as murine or human cytomegalovirusimmediate early promoter), a retrovirus, hepatitis-B virus, and SimianVirus 40 (such as SV 40 early and late promoters) or promoters obtainedfrom heterologous mammalian promoters, such as the actin promoter or animmunoglobulin promoter or heat shock promoters.

Enhancers which can be used are e.g. enhancer sequences known frommammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin)or enhancer from a eukaryotic cell virus. e.g. the SV40 enhancer, thecytomegalovirus early promoter enhancer, the polyoma, and adenovirusenhancers.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e. g., E. coliplasmids col E1, pCR1, pBR322, pcDNA3, pMB9 and their derivatives,plasmids such as RP4; phage DNAs, e. g., the numerous derivatives ofphage X, e. g., NM989, and other phage DNA, e. g., M13 and filamentoussingle stranded phage DNA; yeast plasmids such as the 2μ plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like.

Most preferably the expression vector is pcDNA3.

Another concern of the present invention is to provide a eukaryotic orprokaryotic host cell containing the peptide according to the invention,the isolated and purified nucleic acid sequence of the invention orand/or expression vector described herein.

Transformation or transfection of appropriate eukaryotic or prokaryotichost cells with an expression vector comprising a purified and isolatedDNA sequence according to the invention is accomplished by well knownmethods that typically depend on the type of vector used. With regard tothese methods, see for example, Maniatis et al. 1982, Molecular Cloning,A laboratory Manual, Cold Spring Harbor Laboratory and commerciallyavailable methods. The term “cell transfected” or “cell transformed” or“transfected/transformed cell” means the cell into which theextracellular DNA has been introduced and thus harbours theextracellular DNA. The DNA might be introduced into the cell so that thenucleic acid is replicable either as a chromosomal integrant or as anextra chromosomal element.

The peptide consisting essentially of the N2 sequence of the RasGAPprotein, a fragment thereof, or a variant thereof, optionally conjugatedto an agent which increases the accumulation of the peptide in a cell asdescribed herein are preferably produced, recombinantly, in a cellexpression system.

A wide variety of unicellular host cells are useful in expressing theDNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, AfricanGreen Monkey kidney cells (e. g., COS 1, COS 7, BSC1, BSC40, and BMT10),insect cells (e. g., Sf9), and human cells and plant cells in tissueculture. Preferably, the host cell is a bacterial cell, more preferablyan E. coli cell.

The present invention is also directed to a pharmaceutical compositioncomprising as an active substance a pharmaceutically effective amount ofat least one peptide as described, optionally in combination withpharmaceutically acceptable carriers, diluents and adjuvants.

“A pharmaceutically effective amount” refers to a chemical material orcompound which, when administered to a human or animal organism inducesa detectable pharmacologic and/or physiologic effect.

The respective pharmaceutically effect amount can depend on the specificpatient to be treated, on the disease to be treated and on the method ofadministration. Further, the pharmaceutically effective amount dependson the specific peptide used, especially if the peptide additionallycontains a drug as described or not. The treatment usually comprises amultiple administration of the pharmaceutical composition, usually inintervals of several hours, days or weeks. The pharmaceuticallyeffective amount of a dosage unit of the polypeptide usually is in therange of 0.001 ng to 100 μg per kg of body weight of the patient to betreated.

Preferably, in addition to at least one peptide as described herein, thepharmaceutical composition may contain one or more pharmaceuticallyacceptable carriers, diluents and adjuvants.

Acceptable carriers, diluents and adjuvants which facilitates processingof the active compounds into preparation which can be usedpharmaceutically are non-toxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® orpolyethylene glycol (PEG).

The form of administration of the pharmaceutical composition may besystemic or topical. For example, administration of such a compositionmay be various parenteral routes such as subcutaneous, intravenous,intradermal, intramuscular, intraperitoneal, intranasal, transdermal,buccal routes or via an implanted device, and may also be delivered byperistaltic means.

The pharmaceutical composition comprising a peptide, as describedherein, as an active agent may also be incorporated or impregnated intoa bioabsorbable matrix, with the matrix being administered in the formof a suspension of matrix, a gel or a solid support. In addition thematrix may be comprised of a biopolymer.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma]ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished for example by filtration through sterilefiltration membranes.

It is understood that the suitable dosage of a peptide of the presentinvention will be dependent upon the age, sex, health, and weight of therecipient, kind of concurrent treatment, if any and the nature of theeffect desired.

The appropriate dosage form will depend on the disease, the peptide, andthe mode of administration; possibilities include tablets, capsules,lozenges, dental pastes, suppositories, inhalants, solutions, ointmentsand parenteral depots.

Since amino acid modifications of the amino acids of the peptide arealso encompassed in the present invention, this may be useful forcross-linking the peptide of the invention to a water-insoluble matrixor the other macromolecular carriers, or to improve the solubility,adsorption, and permeability across the blood brain barrier. Suchmodifications are well known in the art and may alternatively eliminateor attenuate any possible undesirable side effect of the peptide and thelike.

While a preferred pharmaceutical composition of the present inventioncomprises a peptide as an active agent, an alternative pharmaceuticalcomposition may contain a purified and isolated nucleic acid sequenceencoding the peptide, as described herein, as an active agent. Thispharmaceutical composition may include either the sole purified andisolated DNA sequence, an expression vector comprising said purified andisolated DNA sequence or a host cell previously transfected ortransformed with an expression vector described herein. In this latterexample, host cell will preferably be isolated from the patient to betreated in order to avoid any antigenicity problem. These gene and celltherapy approaches are especially well suited for patients requiringrepeated administration of the pharmaceutical composition, since thesaid purified and isolated DNA sequence, expression vector or host cellpreviously transfected or transformed with an expression vector can beincorporated into the patient's cell which will then produce the proteinendogenously.

Usually, the pharmaceutical composition as described herein is used forthe treatment or prevention of cancer.

Also encompassed by the present invention is the use of thepharmaceutical composition of the invention, for the preparation of amedicament for the treatment or prevention of cancer.

The term “cancer” refers to or describes the physiological condition inmammals that is typically characterized by unregulated cell growth.

Usually, the cancer to be treated or prevented will be selected from thegroup consisting of carcinoma, lymphoma, blastoma, sarcoma, liposarcoma,neuroendocrine tumor, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, leukemia, lymphoid malignancy, squamous cellcancer, epithelial squamous cell cancer, lung cancer, small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer,esophageal cancer, a tumor of the biliary tract, and head and neckcancer.

Preferably the cancer is mesothelioma, testicular cancer or pancreaticcancer.

The peptide of the invention will generally be used in an amount toachieve the intended purpose. For use to treat or prevent a cancer, thepeptide or the pharmaceutical compositions thereof, is administered orapplied in a therapeutically effective amount. A “therapeuticallyeffective amount” is an amount effective to ameliorate or prevent thesymptoms, or prolong the survival of the subject being treated.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For systemic administration, a therapeutically effective amount or dosecan be estimated initially from in vitro assays. For example, a dose canbe formulated in animal models to achieve a circulating concentrationrange that includes the IC50 as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans.

Initial doses can also be estimated from in vivo data, e.g. animalmodels, using techniques that are well known in the art. One ordinarilyskill in the art could readily optimise administration to humans basedon animal data and will, of course, depend on the subject being treated,on the subject's weight, the severity of the disorder, the manner ofadministration and the judgement of the prescribing physician.

The present disclosure also provides a method of treating or preventingcancer in a subject in need thereof, comprising administering to saidsubject a therapeutically effective amount of the pharmaceuticalcomposition as described herein.

Examples of cancers, which can be treated or prevented, have beendescribed above. Preferably, the cancer is mesothelioma, testicularcancer or pancreatic cancer.

In preferred methods, the subject is a human patient, and theadministered peptide which enhances selectively the ability of at leastone drug to kill cancer cells is the TAT-RasGAP₃₁₇₋₃₂₆ peptide.

Embraced by the scope of the present invention is also a method forenhancing apoptosis selectively in a cancer cell, comprising contactinga cancer cell with at least one peptide of the present invention and adrug.

Also envisioned is a method for selectively killing cancer cellscomprising contacting a cancer cell with at least one peptide of thepresent invention and a drug.

The use of the peptide disclosed herein for enhancing the ability of adrug to kill cells selectively in cancer cells is also envisioned.

A further object of the present invention is to provide a kit fortreating or preventing cancer in a subject, said kit comprising at leastone peptide as described herein optionally with reagents and/orinstructions for use.

Generally, the kit comprises a container and a label or package inserton or associated with the container. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is effective for treating the cancer and mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The label or package insert indicates thatthe composition is used for treating the cancer of choice.

Optionally, the kit further comprises a separate pharmaceutical dosageform comprising an additional anti-cancer agent selected from the groupconsisting of drugs as described above, anti-epidermal growth factorreceptors antibodies, radioimmunotherapeutic agents, and combinationsthereof.

EXAMPLES Example 1 Cells and Transfection

HeLa and MCF-7 cells were maintained in RPMI 1640 (Sigma; cat. no 8758)containing 10% newborn calf serum (Sigma; cat. no N4637) at 37° C. and5% CO₂. U2OS cells were maintained in DMEM (Sigma; cat. no 5796)containing 15% foetal calf serum (Sigma; cat. no F7524) at 37° C. and 5%CO₂. H-Meso-1 cells were maintained in RPMN 1640 containing 10% foetalcalf serum at 37° C. and 5% CO₂. HUV-EC-C cells were maintained in humanendothelial SFM medium (Gibco; cat. no 11111-044) complemented with 10%foetal calf serum, 20 ng/ml of basic fibroblast growth factor (Gibco;cat. no 13256-029), 10 ng/ml of epidermal growth factor (Gibco; cat. no13247-051), 10 μg/ml of fibronectin (Gibco; cat. no 33016-015) at 37° C.and 5% CO₂. HaCat cells were maintained in keratinocyte SFM mediumcontaining epidermal growth factor 1-53 and extract from bovinepituitary gland (Gibco; cat. no 17005-075) at 37° C. and 5% CO₂. HeLacells were transfected as described previously (Yang J.-Y. and WidmannC., Mol. Cell. Biol., 21, 5346, 2001). Genotoxin treatment was performedin 6-well plates. The cells were split the day before the treatment at aconcentration of 2.5×10⁵ cells/well. U2OS cells were transfected in 6well plates using the calcium/phosphate precipitation procedure (JordanM. et al, “Transfecting mammalian cells: optimization of criticalparameters affecting calcium-phosphate precipitate formation” NucleicAcids Res., 24, 596, 1996). Briefly, plasmids were diluted in 90 μl H₂O,mixed with 10 μl CaCl₂ 2.5 M and incubated 10 min at room temperature.Then, 100 μl of HEP solution (280 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, 12mM D-glucose, 50 mM HEPES) was rapidly mixed with the DNA solution,incubated at room temperature for exactly 1 min, and finally transferredto the cell culture medium. After 8 hours at 37° C. and 5% CO₂, themedium was replaced with fresh culture medium and the cells were furtherincubated for 16-24 hours before being analyzed.

Chemicals.

Cisplatin and mitoxantrone were from Sigma (cat. no P4394 and no M6545,respectively). Cisplatin was diluted in DMSO at a final concentration of100 mM and stored at −20° C. Mitoxantrone was diluted in ethanol 100% ata final concentration of 10 mM and stored at −80° C. Adriamycin was fromCalbiochem (cat. no 324380). It was diluted in water at a finalconcentration of 10 mM and stored at −20° C. Hoechst 33342 was fromRoche (cat. no H-1399). It was diluted in water at a final concentrationof 10 mg/ml and stored at 4° C. in the dark.

Peptide Synthesis and Labeling.

The HIV-TAT₄₈₋₅₇ (GRKKRRQRRR) (SEQ ID NO: 15) and TAT-RasGAP₃₁₇₋₃₂₆(GRKKRRQRRRGGWMWVTNLRTD) (SEQ ID NO: 16) peptides were synthesized atthe Institute of Biochemistry, University of Lausanne, Switzerland usingFMOC technology, purified by HPLC and tested by mass spectrometry.

Fluorescein isothiocyanate (FITC)-labeling was performed on the sequenceβ-alanine-GRKKRRQRRRGGWMWVTNLRTD (SEQ ID NO: 16) whose side chainFmoc-protected amino acids were Arg(bpf), Lys(Boc), Gln(Trt), Trp(Boc),Thr(tBu), Asn(Trt), and Asp(OtBu). The peptide was synthesized stepwiseon 0.2 mmol Rink Amide AM resin using Fmoc chemistry. The synthesis wasmonitored by ninhydrin test. After the coupling of β-alanine, the Fmocgroup was removed with 20% piperidine in dimethylformamide (DMF). Atthis stage, a fluorescein group was conjugated to the N-terminus ofpeptide with FITC (5 fold excess over the substitution of the resin in 4ml DMF and 1 ml N-ethyldiisopropylamine) to form thefluorescein-derivated peptide.

Peptides were dissolved in deionised water at a final concentration of 1mM and stored at −20° C. until further use.

Plasmids.

The extension dn3 in the name of a plasmid indicates that the backboneplasmid is the expression vector pcDNA3 (Invitrogen). All the constructswere tagged with the HA sequence (MGYPYDVPDYAS) (SEQ ID NO: 17) at the Namino-terminal end. Plasmid N2.dn3 encodes the human RasGAP fragment N2,plasmids SH2-SH3.dn3 encodes human RasGAP amino acids 158-361, plasmidsSH2.dn3 encodes human RasGAP amino acids 158-277, plasmids SH3.dn3encodes human RasGAP amino acids 279-361. Plasmids IκBαΔN2 encodes aform of IκBα that blocks the activation of NFκB (Yang and Widmann,2002b). Plasmid pEGFP-C1, encoding the GFP protein, was from Clontech.pRL-TK, a vector encoding the Renilla reniformis luciferase, was fromPromega. prLUC is a reporter plasmid bearing the firefly luciferase cDNAunder the control of NFκB-responsive elements (Yang J.-Y. and WidmannC., Mol. Cell. Biol., 21, 5346, 2001).

Apoptosis Measurements.

Apoptosis was determined by scoring the number of cells displayingpycnotic nuclei. Nuclei of live cells were labeled with Hoechst 33342(10 μg/ml final concentration) for about 5 minutes and the cells werethen analyzed (at least 400 cells per condition) using an inverted LeicaDMIRB microscope equipped with fluorescence and transmitted lightoptics. Assessment of apoptosis was performed one day after thetransfection or treatment of the cells. In experiment involvingtransfected cells, pEGFP-C1 was included in the transfection solution tolabel the transfected cells with GFP. In this case, the extent ofapoptosis was assessed in the transfected cells only.

Luciferase Reporter Assay.

Luciferase assay was performed using the Dual-Luciferase® Reporter Assayfrom Promega (cat. no E1910). The cells were lysed from a 6 wells-plateusing 100 μl of PLB lysis buffer provided from the Promega's kit andincubated thirty minutes on ice The lysate was then cleared bycentrifugation at 16'000 g for 15 min. The firefly luciferase activitywas recorded by mixing 20 μl of the lysate with 25 μl of the LARIIReagent and the Renilla luciferase activity was recorded by adding tothe previous mix 25 μl of the Stop & Glo Reagent. For each measurement,light emission was quantified during 12 seconds using a Lumat LB 9501luminometer (Berthold Technologies, Zurich, Switzerland).

Western Blot Analysis

Cells were lysed in lysis buffer (25 mM Hepes, 300 mM NaCl, 1.5 mMMgCl₂, 0.2 mM EDTA, 0.1 mM Na₃VO₄, 1% Triton X100, Complete EDTA-freeProtease inhibitor Cocktail Tablets (Roche; cat. no 1873580). Proteinswere separated on SDS-PAGE and blotted onto nitrocellulose membranes(BioRad; cat. no 162-0115). Thereafter, membranes were blocked with TBS(18 mM HCl, 130 mM NaCl, 20 mM Tris), 5% non-fat dry milk for 30 min atroom temperature and incubated overnight with the appropriate primaryantibody. These antibodies were detected by Alexa Fluor 680 conjugatedsecondary antibodies (Molecular Probes; cat. no A21109) diluted 1:2500in TBS, 5% non-fat dry milk and subsequently visualized with the Odysseyinfrared imaging system (Licor, Homburg, Germany). The primary antibodyagainst phospho-p38 (Cell Signaling Technology; cat no 9211L) wasdiluted 1:500 in 5% BSA in TBS. The primary antibody against phospho-JNK(Cell Signaling Technology; cat n9551L) was diluted 1:1000 in TBS, 5%BSA. Quantitation was performed using the Odyssey infrared imagingsoftware.

Statistical Analysis

All the statistical analyses were performed with Microsoft Excel (XPedition) using the student t-test.

Example 2 RasGAP Fragment N2 Potentiates the Apoptotic Response Inducedby a Variety of Genotoxins

Applicants have recently shown that fragment N2 enhances the ability ofcisplatin to kill the HeLa tumor cell line (Yang J.-Y. and Widmann C.,Mol. Cell. Biol., 21, 5346, 2001). To assess whether fragment N2 couldpotentiate the apoptotic response induced by other genotoxins, HeLacells, expressing or not fragment N2, were subjected to increasingconcentrations of adriamycin and mitoxantrone (and cisplatin ascontrol). FIG. 1 shows that the presence of fragment N2 rendered HeLacells at least 10 times more sensitive than control cells towards thevarious drugs. This result indicates that fragment N2 is a broadspectrum genotoxin sensitizor.

Identification of a Minimal Sequence within Fragment N which Enhancesthe Ability of a Drug to Kill Cancer Cells.

Fragment N2 is a 36 kDa protein, which can make it difficult tosynthesize chemically. Characterization of a shorter sequence that wouldstill bear the genotoxin-sensitizing ability is a critical step in theprocess of developing a therapeutical tool from fragment N2. In order toassess whether such a short sequence could be isolated, we generated aseries of truncated version of fragment N2 (FIG. 2A) and determinedtheir ability to potentiate cisplatin-induced apoptosis in HeLa cells(FIG. 2B). Fragment N2 contains two SH2 domains and one SH3 domain (FIG.2A). We first determined which of these domains contained thepro-apoptotic activity of fragment N2. HeLa cells were transfected withthe plasmids encoding the various SH domains, in the presence or in theabsence of 0.15 μM cisplatin, a concentration at which the difference insensitivity towards the drug between cells expressing or not fragment N2is maximal (see FIG. 1). An empty construct or a construct encoding theSH2 domain did not enhance the ability of cisplatin to kill HeLa cellstransfected with these constructs. In contrast, constructs containingthe SH3 domain enhance the ability of cisplatin to kill cells viapoptosis (FIG. 2B). The inability of the SH2 domain alone to potentiatethe cell death in HeLa cells was not a consequence of reduced proteinexpression because Western blot analysis revealed that the SH2 domainwas expressed as efficiently as the other constructs (data not shown).

Applicants next generated progressive truncations in the SH3 domain inan attempt to identify a minimal genotoxin-sensitizing sequence. Allthese constructs (see FIG. 2A), including the shortest one (317-326)that codes for a 10 amino acid long peptide, potentiated the ability ofcisplatin to kill HeLa cells (FIG. 2B). These results suggest that thecell-death sensitizing property of fragment N2 does not require acomplete SH domain but is mediated by a very short peptidic sequence.

The HIV-TAT₄₈₋₅₇Peptide Conjugated with the 317-326 Rasgap Sequence Actsas Cell Permeable Genotoxin-Sensitizing Peptide.

If a plasmid encoding amino acid 317-326 of RasGAP has the ability toenhance the ability of a drug to kill cancer cells, a synthetic peptidecorresponding to the 316-326 should also display this activity providedthat it can penetrate cells. It has been demonstrated that the additionof a short sequence derived from the HIV TAT protein (HIV-TAT₄₅₋₅₇)allows polypeptides to accumulate efficiently in cells (Schwarze S. R.et al., “In vivo protein transduction: delivery of abiologically activeprotein into the mouse” Science, 285, 1569, 1999). Applicants thereforesynthesized apeptide containing the amino acids 317-326 of RasGAPcovalently bound to the amino acids carrier peptide HV-TAT₄₈₋₅₇. Twoglycine residues were inserted between the TAT and RasGAP sequences asspacer to allow flexibility. As a mean to assess the cellular uptake ofthis peptide (thereafter called TAT-RasGAP₃₁₇₋₃₂₆ peptide), we labeledit with the FITC fluorophore. The labeled peptide was incubated withfour different tumor cell lines: a human adenocarcinoma from cervix(HeLa cells), a human osteosarcoma (U2OS cells), a breast cancer cellline (MCF-7 cells) and a human malignant mesothelomia (H-Meso-1 cells)and two non cancer cell lines (HaCat human skin keratinocyte cell lineand the HUV-EC-C human umbilical endothelial cells). As shown in FIG. 3,the TAT-RasGAP₃₁₇₋₃₂₆ peptide efficiently entered all these cell lines.No difference in translocation of the peptide was noted between the noncancer cells and the cancer cells. We first assessed the sensitivity ofthe cancer cell lines towards increasing concentrations of the threegenotoxins (data not shown). This allowed us to determine the sub-lethalconcentrations of the genotoxins for each cell lines tested.Surprisingly, Applicants have shown that the TAT-RasGAP₃₁₇₋₃₂₆ peptide,but not the control HIV-TAT₄₈₋₅₇ peptide lacking the RasGAP sequences,enhanced the ability of cisplatin, adriamycin, and mitoxantrone to killthe tested cancer cell lines and that did not, or only marginally,induce apoptosis in control conditions. In contrast, the apoptoticresponse induced by cisplatin, adriamycin and mitoxantrone in the twonon cancer lines was unaffected by the presence of the peptides (FIG.4B). We have therefore identified a minimal synthetic peptide able tospecifically enter and enhance the ability of the drugs to kill cancercells.

NFkB and the SAPK Pathways are not Involved in the Properties ofTat-RasGAP₃₁₇₋₃₂₆.

As a Ras modulator, RasGAP could affect some of the Ras-dependentpathways controlling cell death, such as the Ras-PI3K-Akt-NFkB pathway(Datta S. R. et al., “Cellular survival: a play in three Akts” GenesDev., 13, 2905, 1999). We therefore assessed whether modulation of NFkBactivity participate in the enhancing ability mediated byTAT-RasGAP₃₁₇₋₃₂₆. As shown in FIG. 5A, NFkB was activated in U2OS cellsafter a treatment with cisplatin or with TAT-RasGAP₃₁₇₋₃₂₆, but not uponstimulation with the control peptide HWV-TAT₄₈₋₅₇. Incubation of thecells with both cisplatin and TAT-RasGAP₃₁₇₋₃₂₆ resulted in additiveactivation of NFkB (FIG. 5A). The NFkB pathway participates in theinduction of cell survival responses, in many cell types (Van Antwerp D.J. et al., “Suppression of TNF-alpha-induced apoptosis by NF-kappaB”Science, 274, 787, 1996; Beg A. A. and Baltimore D., “An essential rolefor NF-kappaB in preventing TNF-alpha-induced cell death” Science, 274,782, 1996) but can also be required for the induction of apoptoticresponses in some situations (Ryan K. M. et al., “Role of NF-kappaB inp53-mediated programmed cell death” Nature, 404, 892, 2000). Todetermine whether activation of NFkB by TAT-RasGAP₃₁₇₋₃₂₆ was requiredfor the enhancing ability function, a non-degradable form of IkB(IkBαΔN2) was expressed in cells. As expected, this constructefficiently prevented the activation of NFkB by cisplatin andTAT-RasGAP₃₁₇₋₃₂₆ (FIG. 5B). IkBαΔN2 slightly increased the apoptoticresponse mediated by cisplatin (FIG. 5C). The potency ofTAT-RasGAP₃₁₇₋₃₂₆ to enhancing ability of cisplatin to kill U2OS cellsvia apoptosis was however unaffected by the NFkB inhibitor (FIG. 5C).These results indicate that activation of the NFkB pathway is notinvolved in the cell death enhancing ability effect mediated byTAT-RasGAP₃₁₇₋₃₂₆.

The stress-activated protein kinases (SAPKs)—the JNKs and the p38MAPKs—have been implicated in the apoptotic response induced by variousstimuli (Jarpe M. B. et al., Oncogene, 17, 1475, 1998). FIG. 6 showsthat neither the control HIV-TAT₄₈₋₅₇ peptide nor the TAT-RasGAP₃₁₇₋₃₂₆peptide activated these MAPK pathways. These peptides were also not ableto increase the ability of cisplatin to stimulate the JNK or the p38MAPKs (FIG. 6). These results indicate that the stress-activated MAPKpathways are not implicated in the capacity of TAT-RasGAP₃₁₇₋₃₂₆ toenhance the ability of genotoxins to kill cancer cells.

1. A pharmaceutical composition comprising i) at least one peptidefragment of the N2 sequence of the RasGAP protein which comprises theamino acid sequence WXWVTXXRTX (SEQ ID NO:14), wherein said at least onepeptide fragment is less than 90% of the length of said N2 sequence, andwherein X represents an amino acid, or a retro-inverso form of said atleast one peptide fragment, and ii) a genotoxin, wherein said at leastone peptide fragment or the retro-inverso form thereof enhances theability of said genotoxin to kill selectively cancer cells.
 2. Thepharmaceutical composition of claim 1, wherein said at least one peptidefragment comprises at least one amino acid sequence encoded by anucleotide sequences selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
 3. The pharmaceuticalcomposition of claim 1, wherein said at least one peptide fragmentcomprises the amino acid sequence WMWVTNLRTD (SEQ ID NO: 8).
 4. Thepharmaceutical composition of claim 1, wherein said at least one peptidefragment further comprises at least one amino acid in D-form.
 5. Thepharmaceutical composition of claim 4, wherein said D-form amino acidsare in retro-inverso form.
 6. The pharmaceutical composition of claim 1,wherein said at least one peptide fragment is further conjugated to anagent which increases the cell accumulation of said at least one peptidefragment.
 7. The pharmaceutical composition of claim 6, wherein theagent is a cell membrane permeable carrier.
 8. The pharmaceuticalcomposition of claim 7, wherein the cell membrane permeable carrier is apeptide.
 9. The pharmaceutical composition of claim 8, wherein the cellmembrane permeable carrier peptide comprises at least one amino acid inD-form.
 10. The parmaceutical composition of claim 9, wherein saidD-form amino acids are in retro-inverso form.
 11. The pharmaceuticalcomposition of claim 8, wherein the cell membrane permeable carrierpeptide is an arginine rich peptide which is selected from the groupconsisting of an HIV-TAT₄₈₋₅₇ peptide (SEQ ID NO:15), an FHV-coat₃₅₋₄₉peptide, an HTLV-II Rex₄₋₁₆ peptide, and a BMV gag₇₋₂₅ peptide.
 12. Thepharmaceutical composition of claim 8, wherein said at least one peptidefragment comprises at least one amino acid in retro-inverso form. 13.The pharmaceutical composition of claim 1, wherein the genotoxin isselected from the group consisting of an alkylating agent, anantimetabolite, a DNA cutter, a DNA binder, a topoisomerase poison, anda spindle poison.
 14. The pharmaceutical composition of claim 13,wherein said alkylating agent is selected from the group consisting oflomustine, carmustine, streptozocin, mechlorethamine, melphalan, uracilnitrogen mustard, chlorambucil, cyclosphamide, iphosphamide, cisplatin,carboplatin, mitomycin, thiotepa, dacarbazin, procarbazine, hexamethylmelamine, triethylene melamine, busulfan, pipobroman, mitotane, andother platinum compounds.
 15. The pharmaceutical composition of claim 1,wherein said at least one peptide fragment comprises the SH3 domain ofthe N2 sequence, or a part thereof.
 16. The pharmaceutical compositionof claim 1, wherein said at least one peptide fragment comprises atleast one amino acid in retro-inverso form.
 17. The pharmaceuticalcomposition of claim 1, wherein said at least one peptide fragment isless than 60% of the length of said N2 sequence.
 18. The pharmaceuticalcomposition of claim 1, wherein said at least one peptide fragment isless than 30% of the length of said N2 sequence.
 19. A method forenhancing apoptosis selectively in a cancer cell, comprising contactinga cancer cell with a therapeutically effective amount of thepharmaceutical composition of claim
 1. 20. A method for selectivelykilling cancer cells comprising contacting a cancer cell with atherapeutically effective amount of the pharmaceutical composition ofclaim
 1. 21. A pharmaceutical composition comprising i) at least onepeptide fragment of the N2 sequence of the RasGAP protein whichcomprises the amino acid sequence WXWVTXXRTX (SEQ ID NO:14), whereinsaid at least one peptide fragment is less than 90% of the length ofsaid N2 sequence, and wherein X represents an amino acid, or aretro-inverso form of said at least one peptide, and ii) a genotoxin,wherein said genotoxin is a DNA cutter, and wherein said at least onepeptide fragment or the retro-inverso form thereof enhances the abilityof said genotoxin to kill selectively cancer cells.
 22. Thepharmaceutical composition of claim 21, wherein the DNA cutter isbleomycin.
 23. A pharmaceutical composition comprising i) at least onepeptide fragment of the N2 sequence of the RasGAP protein whichcomprises the amino acid sequence WXWVTXXRTX (SEQ ID NO:14), whereinsaid at least one peptide fragment is less than 90% of the length ofsaid N2 sequence, and wherein X represents an amino acid, or aretro-inverso form of said at least one peptide, and ii) a genotoxin,wherein said genotoxin is a topoisomerase poison, and wherein said atleast one peptide fragment or the retro-inverso form therof enhances theability of said genotoxin to kill selectively cancer cells.
 24. Thepharmaceutical composition of claim 23, wherein the toposiomerase poisonis selected from the group consisting of topotecan, irinotecan,camptothecin sodium salt, daorubicin, doxorubicin, idarubicin,mitoxantrone, teniposide, adriamycin, and etoposide.
 25. Apharmaceutical composition comprising i) at least one peptide fragmentof the N2 sequence of the RasGAP protein which comprises the amino acidsequence WXWVTXXRTX (SEQ ID NO: 14), wherein said at least one peptidefragment is less than 90% of the length of said N2 sequence, and whereinX represents an amino acid, or a retro-inverso form of said at least onepeptide, and ii) a genotoxin, wherein said genotoxin is a DNA binder,and wherein said at least one peptide fragment or the retro-inverso formthereof enhances the ability of said genotoxin to kill selectivelycancer cells.
 26. The pharmaceutical composition of claim 25, whereinthe DNA binder is dactinomycin or mithramycin.
 27. A pharmaceuticalcomposition comprising i) at least one peptide fragment of the N2sequence of the RasGAP protein which comprises the amino acid sequenceWXWVTXXRTX (SEQ ID NO:14), wherein said at least one peptide fragment isless than 90% of the length of said N2 sequence, and wherein Xrepresents an amino acid, or a retro-inverso form of said at least onepeptide, and ii) a genotoxin, wherein said genotoxin is a spindlepoison, and wherein said at least one peptide fragment or theretro-inverso form thereof enhances the ability of said genotoxin tokill selectively cancer cells.
 28. The pharmaceutical composition ofclaim 27, wherein the spindle poison is selected from the groupconsisting of vinblastin, vincristin, navelbin, paclitaxel, anddocetaxel.
 29. A pharmaceutical composition comprising i) at least onepeptide fragment of the N2 sequence of the RasGAP protein whichcomprises the amino acid sequence WXWVTXXRTX (SEQ ID NO: 14), whereinsaid at least one peptide fragment is less than 90% of the length ofsaid N2 sequence, and wherein X represents an amino acid, or aretro-inverso form of said at least one peptide, and ii) a genotoxin,wherein said genotoxin is an antimetabolite, and wherein said at leastone peptide fragment or the retro-inverso form thereof enhances theability of said genotoxin to kill selectively cancer cells.
 30. Thepharmaceutical composition of claim 29, wherein the antimetabolite isselected from the group consisting of methotrexate, trimetrexate,pentostatin, cytarabin, ara-CMP, fludarabine phosphate, hydroxyurea,fluorouracyl, floxuridine, chlorodeoxyadenosine, gemcitabine,thioguanine, and 6-mercaptopurine.
 31. A kit for treating cancer in asubject comprising a pharmaceutical composition comprising i) at leastone peptide fragment of the N2 sequence of the RasGAP protein whichcomprises the amino acid sequence WXWVTXXRTX (SEQ ID NO:14), wherein Xrepresents an amino acid, or a retro-inverso form of said at least onepeptide fragment, and ii) a genotoxin, wherein said at least one peptidefragment or the retro-inverso form thereof enhances the ability of saidgenotoxin to kill selectively cancer cells, and instructions for use.32. The kit of claim 31, further comprising a separate pharmaceuticaldosage form including an additional anti-cancer agent selected from thegroup consisting of drugs, anti-epidermal growth factor receptorsantibodies, radioimmunotherapeutic agents, and combinations thereof. 33.The kit of claim 31, wherein said at least one peptide fragment is lessthan 90% of the length of said N2 sequence.
 34. The kit of claim 31,wherein said at least one peptide fragment is less than 60% of thelength of said N2 sequence.
 35. The kit of claim 31, wherein said atleast one peptide fragment is less than 30% of the length of said N2sequence.
 36. A kit for treating cancer in a subject comprising i) atleast one peptide fragment of the N2 sequence of the RasGAP proteinwhich comprises the amino acid sequence WXWVTXXRTX (SEQ ID NO:14),wherein X represents an amino acid; or a retro-inverso form of said atleast one peptide fragment, and ii) a genotoxin, wherein said at leastone peptide fragment or the retro-inverso form thereof enhances theability of said genotoxin to kill selectively cancer cells, andinstructions for use of said at least one peptide fragment or theretro-inverso form thereof and the genotoxin.
 37. The kit of claim 36,wherein said at least one peptide fragment is less than 90% of thelength of said N2 sequence.
 38. The kit of claim 36, wherein said atleast one peptide fragment is less than 60% of the length of said N2sequence.
 39. The kit of claim 36, wherein said at least one peptidefragment is less than 30% of the length of said N2 sequence.
 40. Amethod for enhancing apoptosis in a cancer cell, comprising contactingthe cancer cell with a therapeutically effective amount of i) at leastone peptide fragment of the N2 sequence of the RasGAP protein whichcomprises the amino acid sequence WXWVTXXRTX (SEQ ID NO:14), whereinsaid at least one peptide fragment is less than 90% of the length ofsaid N2 sequence, and wherein X represents an amino acid, or aretro-inverso form of said at least one peptide fragment, and ii) agenotoxin, wherein said at least one peptide fragment or theretro-inverso form thereof enhances the ability of said genotoxin toselectively kill said cancer cell.
 41. The method of claim 40, whereinsaid at least one peptide fragment comprises the SH3 domain of the N2sequence, or a part thereof, or comprises at least one amino acidsequence encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.42. The method of claim 40, wherein the genotoxin is selected from thegroup consisting of an alkylating agent, an antimetabolite, a DNAcutter, a DNA binder, a topoisomerase poison, and a spindle poison. 43.The method of claim 40, wherein the genotoxin is selected from the groupconsisting of cisplatin, mitoxantrone and adriamycin.
 44. The method ofclaim 40, wherein said at least one peptide fragment is less than 60% ofthe length of said N2 sequence.
 45. The method of claim 40, wherein saidat least one peptide fragment is less than 30% of the length of said N2sequence.
 46. A method for enhancing the sensitivity of a cancer cell toa genotoxin comprising contacting the cancer cell with a genotoxin and atherapeutically effective amount of at least one peptide fragment of theN2 sequence of the RasGAP protein which comprises the amino acidsequence WXWVTXXRTX (SEQ ID NO: 14), wherein said at least one peptidefragment is less than 90% of the length of said N2 sequence, and whereinX represents an amino acid, or a retro-inverso form of said at least onepeptide, thereby enhancing the sensitivity of a cancer cell to thegenotoxin.
 47. The method of claim 46, wherein said at least one peptidefragment comprises the SH3 domain of the N2 sequence, or a part thereof,or comprises at least one amino acid sequence encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, and SEQ ID NO:4.
 48. A method of treating cancer in asubject comprising administering to said subject a therapeuticallyeffective amount of i) at least one peptide fragment of the N2 sequenceof the RasGAP protein which comprises the amino acid sequence WXWVTXXRTX(SEQ ID NO: 14), wherein X represents an amino acid, or a retro-inversoform of said at least one peptide, and ii) a genotoxin, wherein said atleast one peptide fragment or the retro-inverso form thereof enhancesthe ability of said genotoxin to kill selectively cancer cells, suchthat said cancer is treated.
 49. The method according to claim 48,wherein the cancer is selected from the group consisting of carcinoma,lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor,mesothelioma, schwanoma, meningioma, adenocarcinoma, melanoma, leukemia,lymphoid malignancy, squamous cell cancer, epithelial squamous cellcancer, lung cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer, atumor of the biliary tract, and head and neck cancer.
 50. The method ofclaim 48, wherein said at least one peptide fragment comprises the SH3domain of the N2 sequence, or a part thereof, or comprises at least oneamino acid sequence encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:4.
 51. The method of claim 48, wherein the genotoxin is selected fromthe group consisting of an alkylating agent, an antimetabolite, a DNAcutter, a DNA binder, a topoisomerase poison, and a spindle poison. 52.The method of claim 48, wherein the genotoxin is selected from the groupconsisting of cisplatin, mitoxantrone and adriamycin.
 53. The method ofclaim 48, wherein said at least one peptide fragment is less than 90% ofthe length of said N2 sequence.
 54. The method of claim 48, wherein saidat least one peptide fragment is less than 60% of the length of said N2sequence.
 55. The method of claim 48, wherein said at least one peptidefragment is less than 30% of the length of said N2 sequence.